The overall goal of this research is to understand the biochemical mechanism of chromosome segregation in mammalian cells. Our efforts have focused on identifying and characterizing the molecular components of the kinetochore, a structure on chromosomes that binds microtubules. This microtubule-kinetochore interaction is essential for the proper alignment and separation of chromosomes during mitosis. Using monoclonal antibodies raised against HeLa chromosome proteins enriched for candidate centromere-kinetochore proteins, we have identified novel proteins that are localized to this region. By analyzing the structures and mitotic functions of these proteins at the biochemical and molecular levels, an understanding of how interactions amongst these proteins lead to chromosome movement will emerge. Our efforts are focused on characterizing CENP-E, a 310 kDa kinetochore associated protein. The restricted expression of CENP-E to late S and G2 phases of the cell cycle and its rapid degradation after mitosis suggests that it is a mitosis-specific protein. The complete cDNA has been isolated and the sequence reveals that it is a novel member of a superfamily of microtubule-based motor proteins called kinesins. This raises the possibility that CENP-E is a mitosis-specific motor that is directly responsible for chromosome segregation or spindle pole separation or both. These proposed functions are consistent with the dynamic properties of CENP-E. It is only transiently associated at the kinetochores during prometaphase and metaphase, and it is redistributed to the spindle midzone at anaphase. This localization pattern predicts that CENP-E is a motor that will move towards the plus-ends of microtubules. When CENP-E is associated at the kinetochore, its plus-ended motor activity would align chromosomes at the metaphase plate. Once released into the spindle midzone, the same motor activity could push apart the overlapping microtubules to separate the spindle poles. Given that the cDNA for CENP-E is cloned, the analysis of CENP-E will focus on identifying and characterizing the domains that are important for its mitotic functions. The motor domain will be analyzed in vitro for microtubule binding and for the direction of movement along microtubules. The importance of CENP-E during mitosis will be examined by introducing antibodies or dominant negative CENP-E mutants into cells to interfere with endogenous CENP-E activities. The molecular basis of the dynamic properties of CENP-E will be examined by mapping the domains that are necessary for CENP-E to localize at the kinetochore and the spindle midzone. The role of phosphorylation in regulating the assembly of CENP-E and these regions will be addressed. To examine the mechanism of assembly of CENP-E at kinetochores, the ability to reconstitute the assembly reaction in an in vitro system will be explored. The biochemical and molecular analysis of this novel mitotic motor should provide a detailed understanding of ne aspect of the molecular mechanism of chromosome movement and spindle separation during mitosis.